DESCRIPTION: The goal of this proposal is to develop effective gene therapy to treat hemophilia A (HemA) by either direct in vivo bone marrow (BM) transfer or by neonatal intravenous delivery using lentiviral (LV) or Foamy viral vectors (FV) expressing human factor VIII (FVIII). Current treatment of HemA patients with repeated FVIII infusions is costly and inconvenient, and ~25% of patients develop anti-FVIII antibodies. Gene therapy that can achieve long-term phenotypic correction without antibody formation is highly desired. BM hematopoietic stem cells (HSC) are ideal targets for gene therapy because they are capable of self-renewal and can differentiate into blood and other types of cells. Intraosseous (IO) delivery of LVs and neonatal delivery of LVs and FVs have been shown to effectively transduce BM cells in mice. These approaches avoid the difficulties encountered by ex vivo gene therapy including loss of stem cell properties and engraftment potential after cell transfer. Furthermore, no in vitr manipulation of stem cells or pre-conditioning of subjects is required. Previously, we have compared IO delivery of LVs utilizing two different promoters driving expression of human FVIII (F8), a ubiquitous human elongation factor-1? (EF1?) promoter (E-F8-LV) and a human megakaryocytic-specific glycoprotein 1b? (GP1b?) promoter (G-F8-LV). Although HSCs can be efficiently transduced by E-F8-LV, robust anti-FVIII immune responses were induced that eliminated functional FVIII in the circulation. In contrast, platelet-specific FVIII expression was achieved following a single IO delivery of G- FVIII-LV, leading to long-term, partial correction of HemA in animals both with and without pre-existing inhibitors1. Our findings suggest that platelets may comprise an ideal vehicle for delivering FVIII: 1) FVIII stored in ?-granules is protected from high-titer neutralizing antibodies; and 2) even relatively small numbers of activated platelets that locally excrete FVIII are sufficient to promote efficient clot formation during bleeding. In the current proposal, we will test the hypothesis that: 1) IO delivery of a further optimized, G-DF8-LV and G-DF8-FV constructs that incorporate a higher-expressing FVIII variant, in combination with agents that suppress the initial innate immune response, will result in more efficient transduction of HSCs and higher levels of FVIII expression - thereby leading to effective treatment of HemA; 2) By comparing neonatal delivery of P- F8-LV directed by a more potent, ubiquitous PGK promoter vs. G-F8-LV (or corresponding FVs), we will determine whether long-term phenotypic correction of HemA can be achieved by neonatal gene therapy; 3) We will establish proof of principal studies for human applications by using the best vectors in humanized mice. At the end of the study, we will be able to decipher the most effective LV or FV system for directing BM HSC gene therapy to treat HemA. Importantly, these studies are specifically designed to pave the way for parallel pre- clinical studies using this delivery platform in HemA dogs and, ultimately, to an optimally designed clinical trial.